Zinc isodecyl phosphorodithioate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24th November 2017 - 1st March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

Adopted March 23rd 2006; Annex 5 corrected 28th July 2011

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All test concentrations and the control
- Sampling method: 2.0 mL was taken at 0, 24 and 72 hours. At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: None, samples were analysed on day of sampling.
- Additional information: Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration (WAF 10 mg/L) but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test solutions were individually prepared loading rates. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for a period of one hour. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning through glass wool and used as test concentrations.
- Controls: Test medium without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture.
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar.

ACCLIMATION
- Acclimation period: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium maintained under the same conditions as the test.
- Culturing media and conditions (same as test or not): Stock culture media different to test, pre-culture media same as test (from 3 days before test start)
- Any deformed or abnormal cells observed: Not stated

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

24 mg CaCO3/L

Test temperature:

23 - 24°C

pH:

8.0 - 8.4

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal: 1.0, 3.2, 10, 32, 100 mg/L
Measured (0h): 0.142, 0.378, 0.558, 0.593, 1.91, 2.69 mg/L
Measured (24h): 0.0167, 0.0528, 0.0379, 0.0380, 0.822, 1.70 mg/L
Measured (72h): 0.0219, 0.122, 0.122, 0.0473, 0.247, 0.974 mg/L
Measured (TWA): 0.029, 0.10, 0.094, 0.078, 0.72, 1.6 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL vessels
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: All-glass, silanized (to reduce adsorption of test item), containing 50 mL of test solution
- Aeration: Not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 1 x 10 4 cells/mL
- Control end cells density: 204.3 x 10 4 cells/mL
- No. of vessels per concentration (replicates): 3 (1 extra replicate of each test group for sampling purposes after 24 hours of exposure)
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable
- Additional information: 1 or 2 replicates of each test concentration without algae

GROWTH MEDIUM
- Standard medium used: yes, M2 medium according to the OECD 201 Guideline
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water
- Culture medium different from test medium: Pre-culture medium same as test medium (from 3 days prior to test start)
- Intervals of water quality measurement: pH measured at start and end of test for all concentrations and control, temperature continuously measured in control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: Not reported
- Adjustment of pH: None
- Photoperiod: Continuous
- Light intensity and quality: 90 to 95 µE.m-2.s-1
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Counted using a microscope and a counting chamber at start of test. Determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm) for remaining observations.
- Chlorophyll measurement: Not measured

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: Yes
- Test concentrations: 1, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: Yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1.6 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Above limit of solubility for test item in test medium

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.094 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks:

and yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.15 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.12 - 0.17 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.25 - 0.36 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.09 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.065 - 0.12 mg/L

Details on results:

Overall, the biological results did not follow a straight dose-response curve at the three highest test concentrations. However, comparing the results of the range-finding test with the results obtained in the final test, the concentrations measured at loading rates of 100 mg/L were comparable between both tests (2.32 and 2.69 mg/L) as were the effects to some degree (11 and 27%). The reason for the stronger inhibition of algal growth at a loading rate of 32 mg/L is unknown.

Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.72 and 1.6 mg/L when compared to the control.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
72-hour ErC50: 0.86 mg/L (95% confidence interval 0.84 - 0.88 mg/L)
Historical range: 0.82 - 2.3 mg/L
72-hour EyC50: 0.34 mg/L (95% confidence interval 0.34 - 0.35 mg/L)
Historical range: 0.2 - 1.1 mg/L

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate or inhibition of yield (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller).

Additionally, the EC10 and EC20 were determined to meet the recommendations as put down in "A Review of Statistical Data Analysis and Experimental Design in OECD Aquatic Toxicology Test Guidelines" by S. Pack, August 1993.

Calculation of ECx values was based on probit analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding average exposure concentrations of the test item.

The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1 Measured Concentrations Versus Nominal Concentrations

Test item WAF loading rate (mg/L)	Measured concentration (mg/L)			TWA (mg/L)
	t=0h	t=24h	t=72 h
1.0	0.142	0.0167	0.0219	0.029
3.2	0.378	0.0528	0.122	0.10
10	0.558	0.0379	0.122	0.094
10 *	0.593	0.0380	0.0473	0.078
32	1.91	0.822	0.247	0.72
100	2.69	1.70	0.974	1.6

* without algae

Table 2 Growth Rate And Percentage Inhibition For The Total Test Period

Test item TWA conc. (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.772	0.0275	6
0.029	1.772	0.0355	3	0.0
0.10	1.726	0.0525	3	2.6
0.094	1.753	0.0228	3	1.1
0.72	0.979	0.0269	3	45*
1.6	1.287	0.0236	3	27*

* effect was statistically significant.

Table 3 Growth Rate And Percentage Inhibition At Different Time Intervals

Test item TWA conc. (mg/L)	n	0 – 24 h		24 – 48 h		48 – 72h
		Mean	%Inhibition	Mean	%Inhibition	Mean	%Inhibition
Control	6	1.950		1.798		1.568
0.029	3	2.100	-7.7	1.671	7.1	1.546	1.4
0.10	3	1.913	1.9	1.778	1.1	1.488	5.1
0.094	3	1.936	0.76	1.781	0.92	1.543	1.6
0.72	3	1.643	15.8	0.979	46	0.314	80
1.6	3	1.894	2.9	1.464	19	0.503	68

Table 4 Yield And Percentage Inhibition For The Total Test Period

Test item TWA conc. (mg/L)	Mean	Std. Dev.	n	%Inhibition
Control	203.3	17.27	6
0.029	203.5	21.25	3	-0.07
0.10	177.9	28.73	3	13
0.094	191.8	12.95	3	5.7
0.72	17.9	1.49	3	91*
1.6	46.5	3.35	3	77*

* effect was statistically significant.

Table 5 Effect Parameters

	Parameter (mg/L)	NOEC	EC10	EC20	EC50
Growth rate	Value	0.094	0.15	0.42	>1.6
	lower 95%-cl		0.12	0.37
	upper 95%-cl		0.17	0.47
Yield	Value	0.094	0.090	0.14	0.30
	lower 95%-cl		0.065	0.11	0.25
	upper 95%-cl		0.12	0.17	0.36

cl: confidence limit

Table 6 Mean Cell Densities (x10⁴Cells/mL) During the Combined Limit/Range-Finding Test

Time (h)	Test item; WAF loading rate (mg/L)
	Control	1.0	10	100
0	1.0	1.0	1.0	1.0
24	7.5	n.d.	n.d.	7.9
48	53.7	n.d.	n.d.	41.5
72	243.8	264.6	145.6	136.3

n.d.: not determined.

Table 7 Percentage Inhibition of Growth Rate During the Combined Limit/Range-Finding Test

Test item WAF loading rate (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	1.832	0.0203	6
1.0	1.859	0.0241	3	-1.5
10	1.659	0.0417	3	9.5
100	1.637	0.0256	6	11

Table 8 Percentage Inhibition of Yield During the Combined Limit/Range-Finding Test

Test item WAF loading rate (mg/L)	Mean	Std. Dev.	n	% Inhibition
Control	242.8	15.12	6
1.0	263.6	18.73	3	-8.6
10	144.6	18.62	3	40
100	135.3	11.01	6	44

Validity criteria fulfilled:

yes

Remarks:

Cell density increased by 204 in the control; mean coefficient of variation for section by section specific growth rates in control was 11%, coefficient of variation for average specific growth rates in replicate controls was 1.6%

Conclusions:

The 72-hour EC50 was >1.6 mg/L, NOEC was 0.094 mg/L and EC10 was 0.15 mg/L, based on growth rate.

Executive summary:

Augusiak (2018) is a GLP-compliant study following OECD guideline 201. Test solutions were prepared as water accommodated fractions (WAF) based on the OECD guidance for difficult to test substances. Nominal loading rates of 1, 3.2, 10, 32 and 100 mg/L were used, corresponding to measured concentrations of 0.029, 0.1, 0.094, 0.72 and 1.6 mg/L (time weighted average). Test concentrations for the main study were determined based on results from a range-finding study. A 72 -hour exposure was conducted, with analysis of test concentrations at 0, 24 and 72 hours. At the start of the test, the actual test concentrations increased from 0.14 to 2.7 mg/L with increasing loading rate. At the end of the exposure period, the measured concentrations had decreased to 13-36% of the initial concentrations. Three replicates were used for each test concentration and 6 replicates for the control. The initial algal cell density was 1 x 10⁴ cells/mL. All validity criteria for the study were met. The 72-hour EC50 was >1.6 mg/L, NOEC was 0.094 mg/L and EC10 was 0.15 mg/L, based on growth rate. The 72 -hour EC50 was 0.3 mg/L, NOEC was 0.094 mg/L and EC10 was 0.09 mg/L, based on yield.

Description of key information

The 72-hour EC50 was >1.6 mg/L, NOEC was 0.094 mg/L and EC10 was 0.15 mg/L, based on growth rate.

Additional information

Augusiak (2018) is a GLP-compliant report following OECD guideline 201. Test solutions were prepared as water accommodated fractions (WAF) based on the OECD guidance for difficult to test substances. Nominal loading rates of 1, 3.2, 10, 32 and 100 mg/L were used, corresponding to measured concentrations of 0.029, 0.1, 0.094, 0.72 and 1.6 mg/L (time weighted average). Test concentrations for the main study were determined based on results from a range-finding study. A 72 -hour exposure was conducted, with analysis of test concentrations at 0, 24 and 72 hours. At the start of the test, the actual test concentrations increased from 0.14 to 2.7 mg/L with increasing loading rate. At the end of the exposure period, the measured concentrations had decreased to 13-36% of the initial concentrations. Three replicates were used for each test concentration and 6 replicates for the control. The initial algal cell density was 1 x 10⁴ cells/mL. All validity criteria for the study were met. The 72-hour EC50 was >1.6 mg/L, NOEC was 0.094 mg/L and EC10 was 0.15 mg/L, based on growth rate. The 72 -hour EC50 was 0.3 mg/L, NOEC was 0.094 mg/L and EC10 was 0.09 mg/L, based on yield.